SOX18-enforced expression diverts hemogenic endothelium-derived progenitors from T towards NK lymphoid pathways

Summary Hemogenic endothelium (HE) is the main source of blood cells in the embryo. To improve blood manufacturing from human pluripotent stem cells (hPSCs), it is essential to define the molecular determinants that enhance HE specification and promote development of the desired blood lineage from HE. Here, using SOX18-inducible hPSCs, we revealed that SOX18 forced expression at the mesodermal stage, in contrast to its homolog SOX17, has minimal effects on arterial specification of HE, expression of HOXA genes and lymphoid differentiation. However, forced expression of SOX18 in HE during endothelial-to-hematopoietic transition (EHT) greatly increases NK versus T cell lineage commitment of hematopoietic progenitors (HPs) arising from HE predominantly expanding CD34+CD43+CD235a/CD41a−CD45− multipotent HPs and altering the expression of genes related to T cell and Toll-like receptor signaling. These studies improve our understanding of lymphoid cell specification during EHT and provide a new tool for enhancing NK cell production from hPSCs for immunotherapies.


INTRODUCTION
The SOXF family transcription factors, SOX7, SOX17 and SOX18, have been recognized as critical regulators of angiogenesis, cardiovascular and hematopoietic development. [1][2][3][4][5][6][7][8] SOXF factors are expressed in hemogenic endothelium (HE) and clusters of hematopoietic cells emerging from HE. 3,4,6,9,10 However, their expression is transient and not detected in terminally differentiated lymphoid and myeloid cells. Murine embryonic studies have shown that Sox7 is required for the formation of the earliest multipotent HPs with erythro-myeloid potential. 4 Forced expression of Sox7 in cells from E7.5 mouse embryo or from in vitro differentiated mouse embryonic stem cells (ESCs) promotes self-renewal of early CD41 + hemogenic progenitors with erythro-myeloid potential and blocks their differentiation. 4,11 A similar phenotype was observed following overexpression of Sox18 in in vitro differentiated mouse ESCs. 3 In contrast, Sox17 is required for arterial specification, 12 establishing the definitive, but not primitive, hematopoietic program, 10 and maintaining intra-aortic hematopoietic clusters and fetal liver hematopoietic stem cells (HSCs). [5][6][7] Using hPSCs, we demonstrated that SOX17 is a master regulator of HOXA and arterial programs in HE, and is required for the specification of HE with robust lympho-myeloid potential and DLL4 + CXCR4 + phenotype resembling arterial HE at sites of HSC emergence. 13 With activation of NOTCH signaling, SOX17 directly activates CDX2 expression leading to the upregulation of the HOXA cluster genes. 13 Here, we investigated SOX18 effects on hematopoietic development from hPSCs. We demonstrated that enforced SOX18 expression has a limited effect on specification and diversification of HE but significantly affects NK versus T cell commitment when overexpressed in HE and during EHT. Specifically, SOX18 overexpression expands and greatly enhances NK cell potential of CD34 + CD43 + CD235a/CD41a À CD45 À multipotent hematopoietic progenitors (HPs), predominantly leading to the altered expression of genes related to T cell and Toll-like receptor signaling.

SOX18-enforced expression enhances production of erythro-myeloid progenitors
To determine the impact of SOX18 overexpression on hematopoietic development in humans, we generated H1 hESCs carrying doxycycline (DOX)-inducible SOX18-P2A-Venus ( Figure S1) and differentiated these cells iScience Article under a chemically defined culture system in which all stages of hematopoietic development are temporally, phenotypically, and functionally defined ( Figure 1A). In this differentiation system, the most primitive hemogenic cells with FGF2-dependent hemangioblast colony-forming cell (HB-CFCs) potential are detected on day 3 (D3) of differentiation. [14][15][16] The first immature/primordial VEC + CD43 À CD73 -NOTCH1 + HE cells expressing high levels of HAND1 mesodermal gene and lacking of arterial and venous gene expression arise on D4 (D4 HE). Subsequently on D5, HE specifies into DLL4 + CXCR4 +/À arterial-type HE and DLL4non-arterial-type HE ( Figure 1A). 14,16-18 D5 DLL4 + CXCR4 + HE is highly enriched in T lymphoid and multipotential myeloid progenitors and expresses the highest levels of HOXA and arterial genes, including SOX17 and NOTCH4, as compare to other DLL4 + and DLL4 -HE populations. 13 All hematopoietic progenitors derived from hPSC cultures on D8 of differentiation can be identified by CD43 expression. 19,20 D8 CD34 + CD43 + HPs are composed of at least three major subpopulations: (1) CD235a + CD41a + CD45 À progenitors enriched in erythro-megakaryocytic cells, (2) CD41a lo CD235a +/À CD45 + progenitors with erythro-myeloid potential, and (3) CD235a À CD41a À CD43 + CD45 +/À multilineage progenitors that are lacking lineage markers, and display CD90 + CD38 À CD45RA À phenotype [19][20][21][22] typical for human hematopoietic stem/progenitor cells (HPSCs). 23 In addition, it has been shown that SOX18 expression is initiated on D4 differentiation in HE and remains present in D5 HE and D8 HPs. 16,18 To determine the stages of hematopoietic development sensitive to SOX18 modulation and the optimal duration of SOX18 overexpression to achieve a maximal effect on hematopoietic output, we treated hESC differentiation cultures with DOX at different time points and analyzed the phenotype and CFC potential of hematopoietic cells collected on D8 of differentiation (Figure 1B). As shown in Figures 1C and 1D, treatment of cultures with DOX from D2 through D8 (DOX2-8) resulted in the highest percentages of CD34 + CD43 + HPs. We also noted that this treatment increased the proportion of CD235a/CD41a À CD45 À progenitors (D8 P1 population) within CD34 + CD43 + population and decreased a proportion of CD235a/CD41a + CD45 + progenitors (D8 P3 population). Although a similar effect on subset composition within CD34 + CD43 + progenitors was observed in DOX6-8, DOX treatments at these stages of differentiation had a minimal effect on the percentages of CD34 + CD43 + cells. In contrast, earlier and shorter treatments (DOX2-4) mildly increased CD34 + CD43 + cells, but had a minimal effect on their composition.
To visualize the in-depth phenotype of D8 hematopoietic cells from No DOX and DOX2-8 cultures, CD43 + gated cells were analyzed using the t-distributed stochastic neighbor embedding algorithm (tSNE) which showed distinct single cell deposition between No DOX and DOX2-8 ( Figure S2A). tSNE analysis also highlighted a suppression of CD45 + populations and enrichment of CD45 À and CD235a/41a À populations following DOX treatment. In addition, DOX treatment reduced populations with CD235a/ 41a hi/med CD34phenotype ( Figures S2B and S2C) corresponding to more mature erythro-myeloid cells. This observation is consistent with prior findings in murine system, which demonstrated a blocking effect of Sox18 on differentiation of ESC-and yolk sac-derived HPs. 3 Analysis of CFCs revealed the most substantial effect of SOX18 upregulation in DOX2-6 and DOX2-8 cultures where we observed a significant increase in a frequency of GM-and E-CFCs ( Figure 1E). Furthermore, we found that DOX treated cultures had a higher megakaryocytic differentiation potential than non-treated

SOX18-enforced expression promotes HB-CFCs but has a limited effect on specification of hemogenic endothelium formation
To determine stages of hematopoiesis mostly affected by SOX18 overexpression, we analyzed the effect of DOX treatment on the formation of the HB colonies and HE, including arterial HE specification. We found that enforced expression of SOX18 on D2 of differentiation resulted in almost 3-fold increase in the numbers of HB colonies ( Figure S3C). However, SOX18 overexpression had a limited effect on HE formation and arterial HE specification on D4 and D5 of differentiation (Figures 2A-2F). Although we noted a slight increase in the proportion of VE-cadherin + (VEC + ) endothelial cells in DOX-treated cultures, no significant differences were observed in the proportion of DLL4 + CXCR4 +/À arterial HE in DOX and No DOX cultures. We also observed the formation of VEC + DLL4 À CXCR4 + population in DOX-treated D4 cultures ( Figure 2A). However, transcriptional profiling of DLL4 À CXCR4 + and DLL4 À CXCR4 -D4 VEC + populations revealed no differentially expressed genes, including arterial genes (see ''molecular characterization of SOX18-induced changes in HE and blood cells'' section below).
To assess SOX18 effect on HE, we isolated D4 HE generated in No DOX and DOX conditions and cultured on OP9-DLL4 in presence or absence of DOX ( Figure 3A). Analysis of CFC potential revealed that treatment of HE with DOX (DOX4-8) or continuous treatment with DOX throughout culture (DOX2-8) significantly increased CFC formation whereas HE from primary differentiation cultures pretreated with DOX (DOX2-4) without additional DOX treatment during co-cultures with OP9-DLL4 generated fewer CFCs as compared to No DOX controls ( Figure 3B).
Analysis of T cell potential revealed that HE from DOX2-4 treated cultures demonstrated decreased CD5 + CD7 + and CD4 + CD8 + T cell output accompanied by increased in CD7 + CD5 À cells. This effect was more pronounced when DOX treatment was initiated on D4 in co-culture of HE, or in DOX2-8 treatment cultures ( Figures 3C-3E). In contrast, treatment of HE in OP9-DLL4 co-cultures markedly increased NK cell potential of D8 HPs, whereas D2-4 DOX treatment of primary differentiation cultures had no effect on NK cell output from HE ( Figures 3F and 3G). These findings suggest that SOX18 overexpression predominantly affects lymphoid specification from HE by shifting the balance of NK versus T lymphocyte differentiation potential.
SOX18-enforced expression promotes expansion of CD34 + CD43 + CD235a/CD41a À CD45 À (D8 P1) population enriched in NK cell potential As shown in Figures 1C and 1D, D2-D8 SOX18 overexpression significantly promoted development of CD235a/CD41a À CD45 À (D8 P1) populations and inhibited development of CD45 + populations, including CD235A/CD41a + (D8 P3) and CD235a/CD41a À (D8 P4) populations within CD34 + CD43 + HPs. To define cell populations enriched in NK cells following DOX treatment, we isolated major populations of HPs formed on D8 of culture and assessed their NK cell potential ( Figure 4A). As shown in Figures 4B and 4C, in control cultures, NK cell potential was mostly detected in D8 P1 and P4 populations, while P2 population failed to produce NK cells. Although CD235a/CD41a + CD45 + (D8 P3) cells were capable of producing NK cells (Figure S4A), the total NK cell output from this subset was negligible, as compared to the P1 and P4 populations ( Figure 4C). Owing to dramatic inhibition of CD45 + cell development in DOX treated cultures, we evaluated the NK cell potential of only two major populations: (1)   iScience Article expanded following DOX treatment and (2) the D8 P2 population. These studies revealed that, in DOXtreated cultures, NK cell potential was detected in P1 and P2 populations ( Figure 4B). However, total NK cell generation from P2 population was negligible ( Figure 4C). Thus, we concluded that in DOX-treated cultures NK cell potential is mostly restricted to the D8 P1 population and that this population possesses much stronger NK cell differentiation potential as compared to the NK-producing populations in the control No DOX cultures. Overall, HPs collected from DOX2-8 treated cultures generate up to 5-fold more NK cells as compared to the control ( Figure 4D). Moreover, kinetic analysis of NK differentiation cultures revealed a more rapid and longer expansion of NK cells generated from HPs in DOX-treated differentiation cultures ( Figure 4E). Functional assessment of NK cells revealed no substantial differences in cytotoxic potential, IFNg production or degranulation response against K562 cells of NK cells generated from DOX and No DOX conditions ( Figures 4F-4H). Overall, these studies indicate that enforced SOX18 expression predominantly expands CD34 + CD43 + CD235a/CD41a À CD45 À (D8 P1) HP population with superior NK cell potential.

Molecular characterization of SOX18-induced changes in HE and blood cells
To define changes in transcriptional program induced by forced SOX18 expression, we performed RNAseq analysis of D4 HE, D8 CD34 + CD43 + subsets and NK cells generated from No DOX and DOX-treated cultures ( Figure 5A). We found only 15 differentially expressed genes (DEGs) in the D4 DLL4 À CXCR4 -VEC + population (D4 P1 population) following D2-4 DOX treatment ( Figures 5B and 5C and Data S1), suggesting minimal effect of SOX18 on D4 HE. In contrast to our prior findings of D2-4 SOX17 overexpression, 13 RNA-seq analysis of SOX18 D4 HE from No DOX and DOX cultures showed no significant increase in expression of HOXA genes, CDX2, or genes involved in NOTCH signaling pathways. These findings were confirmed using qPCR analysis of selected genes in D4 CD31 + HE cells isolated from control and D2-4 DOX-treated differentiation cultures of iSOX17 13 and iSOX18 hPSCs ( Figure 5D). Because our flow cytometric analysis showed that SOX18 overexpression induces DLL4 À CXCR4 + subset within D4 HE (Figures 2A and 5A), we analyzed DEGs in DLL4 À CXCR4 + (D4 P1) and DLL4 À CXCR4 -(D4 P2) populations from DOX-treated cultures. These studies revealed no DEGs in these subsets, including expression of arterial genes, thus suggesting that CXCR4 expression in D4 HE without co-expression of DLL4 does not reflect activation of arterial program.
The most significant changes in transcriptional programs were observed in the D8 P1 multipotent HP population and in CD56 + NK cells which showed 209 and 811 DEGs in their corresponding DOX treated and untreated cultures ( Figure 5B and Data S1). Gene set enrichment analysis (GSEA) in D8 P1 cells revealed enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG) categories related to T cell receptor (TCR) and Toll-like receptor (TLR) signaling pathways ( Figure 5E), suggesting that SOX18 overexpression has a major effect on establishing T lymphoid transcriptional program in the D8 P1 multipotent HPs arising from HE. As shown in Figure 5F, downregulated genes in these categories included CD3 complex genes, CD4, AP1 complex genes JUN and FOS, PI3K signaling genes and the majority of TLR genes, whereas upregulated genes included IFNG, TICAM2, TLR5 and TLR9. D8 P2 population in DOX+ and DOX-conditions showed only 79 DEGs enriched in extracellular matrix (ECM) receptor interactions KEGG categories (Figure 5E and Data S1). GSEA in NK cells generated from DOX treated and untreated cultures demonstrated enrichment in KEGG categories related to glyoxylate and dicarboxylate metabolism, one carbon pool by folate, and PPAR signaling pathway ( Figure 5E and Data S1). However, no differential expression of NK cell activating receptors KLRK1, NCR2, NCR1 or key NK cell transcription factors EOMES and TBX21 was observed between NK cells from DOX-treated and untreated cultures (Data S1). These findings indicate that overexpression of SOX18 during EHT has a major impact on the transcriptional program regulating metabolism of NK cells.
To determine whether SOX18 overexpression affects proliferation and apoptosis in major cell subsets, we performed gene sets enrichment analysis in GO categories related to ''Regulation of Cell Population iScience Article Proliferation'' and ''Apoptosis''. We found that the first GO gene set was significantly altered in D4 and D8 P1 subsets and NK cells in DOX+ versus No DOX comparisons, whereas apoptotic genes were mostly affected in D8 P1 subset ( Figures S5A and S5B). To confirm the effect of SOX18 on cell proliferation, we performed cell-cycle analysis of D5 differentiated cells using bromodeoxyuridine (BrdU). Consistent with gene expression analysis, SOX18 overexpression led to a significant cell-cycle shift from G0/G1 to S phases in HE cells and emerging CD43 + HPs ( Figures S5A and S5B).

DISCUSSION
Previous studies using inducible murine ESCs or embryos revealed that SOX18 overexpression during the early stages of hematopoietic development enhances the development of blast colonies composed of immature HPs. 3 Herein, we investigated how SOX18 overexpression affects specification and diversification of HE from hPSCs. Using an inducible hPSC line, we demonstrated that SOX18 overexpression during the mesodermal stage of differentiation has little effect on HE specification, including establishment of arterial phenotype. However, SOX18 exerted a profound effect on the development of NK cells versus T cells when upregulated in HE and during EHT. We also inferred that SOX18-enforced expression promotes the development of HB-CFCs and erythro-myeloid progenitors, including megakaryocytes. Overexpression of SOX18 downregulates expression of CD45 within hPSC-derived CD34 + CD43 + HPs and promotes generation of CD34 + CD43 + CD235a/CD41a À CD45 À cells (D8 P1 population) enriched in NK cell potential. Molecular profiling of D8 P1 populations generated with and without DOX treatment showed that SOX18 overexpression has the most significant impact on genes involved in TCR and TLR signaling pathways. These findings suggest that SOX18 has a major effect on specification and lineage commitment of HPs arising from HE.
In addition to their role in hematopoietic development, SOXF group factors including SOX17 and SOX18 are involved in cardiovascular development, where they function in redundant mode. 1,24,25 Our current SOX18 and previous SOX17 studies 13 suggest that the function of these transcription factors in human hematopoietic development is likely distinct and non-redundant. Although SOX17 has the most profound effect on specification of DLL4 + CXCR4 + arterial HE with T lymphoid potential, 13 SOX18 demonstrated no effect on development and formation of arterial HE. However, SOX18 affected NK versus T lymphoid potential of hemogenic progenitors when overexpressed in already established HE. Molecular profiling studies revealed that in contrast to SOX17, 13 SOX18 overexpression at the mesodermal stage of development caused very limited changes in the gene expression profile of HE and did not affect arterial, NOTCH signaling, CDX2 or HOXA cluster gene expression in HE. In contrast, SOX18 overexpression during EHT caused the most significant changes in transcriptional program of multipotent HPs arising from HE, mostly affecting genes involved in TCR and TLR signaling.
Hematopoietic development in the vertebrate embryo occurs in multiple waves. The first transient wave of hematopoiesis takes place from HB in the yolk sac blood islands giving rise to primitive erythroid, megakaryocytic and macrophage cells that are different from their corresponding adult counterparts. In contrast to the first wave of primitive hematopoiesis lacking of lymphoid and granulocytic potential, subsequent waves of definitive hematopoiesis produce the entire spectrum of adult-type erythro-myeloid progenitors (EMPs), lymphomyeloid progenitors, and finally HSCs with the capacity for long-term repopulation of an adult recipient. [26][27][28][29] However, it became evident that EMPs can also produce NK cells and g/d T cells arising from CD7 + CD5 À lymphoid progenitors. [30][31][32] Recent studies demonstrated that in hPSC cultures, NK cells originate from at least two independent CD34 + hemogenic progenitors which can be discriminated based on HOXA expression. HOXA low/À CD34 + progenitors produce NK cells enriched in genes associated with innate immune response, expressing higher levels of CD16 and displaying more robust cytotoxic and degranulation potential, whereas HOXA + CD34 + progenitors generate NK cells enriched in genes associated with inflammatory responses and greater IFNg production. 32 Our studies revealed that SOX18-enforced expression had no effect on proportion of CD16 + or CD94 + cells within CD56 + NK cell iScience Article population and did not affect cytotoxic potential of NK cells against K562 targets or expression of genes associated with innate immune response or inflammation. These observations suggest that SOX18 does not cause a preferential expansion of NK population with distinct degranulation versus pro-inflammatory responses. iScience Article NK cell therapies combined with genetic engineering technologies have emerged as increasingly powerful tools for immunotherapies of cancers. The use of hPSCs as an unlimited source of NK cells can further expand the applicability of cellular immunotherapies by offering ''off-the-shelf'' therapeutic products to fit specific clinical needs for a broad group of patients. 33,34 Our finding that SOX18 promotes development of HPs with superior NK cell potential, whereas inhibiting T cell development, will advance our comprehension of molecular network that regulates specification of lymphoid cells from HE. This understanding will help to improve the scalability of NK cell production from hPSCs for immunotherapies.

Limitations of the study
Although these studies revealed a distinct and contrasting effect of SOX18 overexpression, as compared to SOX17 overexpression, on specification and gene expression in HE, HPs, and lymphoid cells from hPSCs, the molecular mechanisms mediating this effect remains to be addressed. We have shown an increased number of CD34 + CD43 + CD235a/CD41a À CD45 À HPs enriched in NK cell potential following SOX18 overexpression. Subsequent studies will define whether SOX18 is required for development of this population or NK cell specification.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

INCLUSION AND DIVERSITY
One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in their field of research or within their geographical location. While citing references scientifically relevant for this work, we also actively worked to promote gender balance in our reference list.  Figure S4B).

Apoptosis and cell cycle analysis
Apoptosis was detected by flow cytometry using Annexin V (BD). For cell-cycle analysis, D5 cells were incubated in culture medium with BrdU (10 mM, BD Pharmingen) for 2 hours and stained with antibodies. For BrdU detection, the BrdU flow kit with 7 AAD was used and performed per the manufacturer's instructions. Fluorescent reagents used for analysis, cell viability, apoptosis, and proliferation are listed in key resources table.

Real time qPCR
RNA was extracted from D4 CD31 + cells isolated from control and DOX-treated cultures of iSOX18 of iSOX17 13 hPSCs using the RNeasy Plus MicroKit (QIAGEN). RNA was reverse-transcribed into cDNA using random hexamer primers (QIAGEN) with SMART MMLV reverse transcriptase (TaKaRa). qPCR was conducted using TB Green Advantage qPCR Premix (TaKaRa). RPL13A was used as the reference gene to normalize the data. Primer sequences are listed in key resources table.

RNA-seq
One hundred nanograms of total RNA was used to prepare sequencing libraries following the Ligation Mediated Sequencing (LM-Seq) protocol 41

Western Blot
For Western Blot experiment, iSOX18 hPSCs were cultured with (2 mg/ml) and without DOX for 24 hours and harvested. In a similar manner, total cells from iSOX18 hPSC differentiation cultures with (D2-D5) and without DOX were collected at day 5 of differentiation. The cells were lysed using Pierce IP lysis buffer with Pierce protease inhibitors. For cell lysate analysis, protein levels were quantified using the Pierce BCA Assay kit (Thermo Fisher, Waltham, MA) and normalized to 8 mg of total protein (depending on the individual blot) prior to running on pre-cast 4-12% gradient SDS-PAGE gels and subsequent transfer to PVDF membranes using Bio-Rad Trans-Blot Turbo Transfer System. The membrane was blocked with 5% BSA (Fisher Scientific, BP1600-100) and 5% Difco TM Skim Milk (BD, 232100) in TBST (1%) for human SOX18 antibody (R&D Systems, 1:1000) and anti-GAPDH (Santa Cruz Biotechnology, 1:5000) probing respectively. The membranes were incubated with primary antibodies overnight at 4 C after blocking with mild agitation. The membranes were blotted with their corresponding HRP-linked secondary antibodies at room temperature for one hour. The antibodies were diluted in 1% BSA and 1% milk in TBST for SOX18 and GAPDH detection respectively. 1% TBST was used to wash the membranes for three times at 5 minutes intervals. Sheep and rabbit HRP-linked secondary antibodies were purchased from R&D Systems and Santa Cruz Biotechnology. Images were collected using Bio-Rad ChemiDoc TM XRS+.

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